The present invention relates generally to compounds and methods for identifying polymorphism in a cellular receptor, and more particularly, to compounds and methods for identifying and typing single nucleotide polymorphisms that code for a complement receptor and applying these polymorphisms to delineation of disease susceptibility and severity.
The complement system involves a group of proteins that play a role in host defense against infection. Complement is active in immune defenses, especially antibody mediated events by way of the xe2x80x9cclassical pathway.xe2x80x9dComplement response to an invasion by a foreign particle can also embody an antibody independent mechanism which is known as the xe2x80x9calternative pathway.xe2x80x9d
As part of the body""s natural defenses, the complement system operates as a biological cascade in which one component activates successive components. Activation is usually by way of a proteolytic cleavage event. The complement system functions to promote the inflammatory response, to modify the membranes of infectious organisms, and also to identify pathogenic material for removal.
The inflammatory response is triggered by way of the cascade, in which low molecular weight peptides are cleaved from complement proteins. The resulting anaphylatoxins and chemotactic factors attract leukocytes and modify vascular permeability.
The attachment of complement proteins, C3b/C4b to microbial membranes or immune complexes, a process commonly known as opsonization, facilitates the binding of opsonized material to cell receptors. By modifying the membranes of a microbe, complement also participates directly in microbial destruction.
Immune complex opsonization enables the process in which specific receptors on a cell bind complement components. In some cases this binding process leads to phagocytosis of the complement bound antigen particles. The primary opsonins of the complement system are C3b and C4b which are activated by cleavage of fragments C3 and C4. C3b and C4b are capable of covalently binding to foreign antigen particles. The resulting C3b/C4b coded complexes are ligands for the C3b/C4b receptor on human peripheral blood cells. The C3b/C4b receptor is also known as complement receptor 1 (CR1).
Receptors for complement proteins function to bind complement coated antigens. CR1 is present on most peripheral blood cells, including erythrocytes, B lymphocytes, granulocytes, monocytes, neutrophils and some T cells. Erythrocyte CR1 upon binding the C3b/C4b immune complex conveys the immune complex to the liver or spleen where the ligand is transferred to hepatic macrophages for internalization and disposal. As a result, CR1 plays a role in neutralizing complement activated immune complexes within the body. Thus, defects in this receptor are associated with impaired phagocytosis and can lead to enhanced destructive processes such as joint destruction in rheumatoid arthritis, and impaired host defense against infection.
The role of complement receptor 2 (CR2) is currently not fully understood. Nonetheless, CR2 binds activated complement components and is operative in response to foreign particles.
Autoimmune diseases are often characterized by abnormal deposits of complement fixing immune complexes. Such deposits may result from excessive immune complex formation, inappropriate antibody production illustratively including IgG, IgM and IgA, complement deficiencies or receptor anomalies. Receptors that are incapable of binding and/or releasing immune complex or that are underexpressed on the cell surface would result in incomplete removal of immune complexes thereby resulting in deposition.
Such abnormal deposits are associated with a variety of diseases including several types of human glomerulonephritis. In particular, systemic lupus erythematosus (SLE) is associated with glomerular deposits of early complement cascade components such as C1q, C4 and C2. Deposition of these components is suggestive of the classical pathway of complement activation in this disease. Other types of glomerulonephritis such as IgA neuropathy, bacterial infections and membranoproliferative glomerulonephritis are typically associated with glomerular deposits of C3 and properdin, thus suggesting alternative pathway dysfunction in these diseases. The CR1 receptor in humans and primates is found on most types of peripheral blood leukocytes and erythrocytes, however not on platelets. Due to the large fraction of erythrocytes in blood, a great majority of all CR1 in peripheral blood is found on erythrocytes. Perhaps greater than 90% of all CR1 is found on erythrocytes. The characterization of CR1 has previously failed to make a definitive correlation between CR1 structure and immunological disease severity.
CR1 is a single polypeptide chain that exhibits a size polymorphism derived from four codominant inherited alleles. The alleles are: type A (220 kD), type B (250 kD), type C (190 kD) and type D (280 kD). These four alleles result in ten phenotypes of CR1 within the human population which have varying numbers of C3b binding sites. Fluctuations in erythrocyte CR1 expression associated with SLE progression suggests that genetic predisposition as to size polymorphism is not a controlling factor in SLE (Please fill in reference prop #110, now 1). While SLE is an important autoimmune disease, fluctuating levels of CR1 are also associated with diseases including hemolytic anemias, AIDS, rheumatoid arthritis, Sjogren""s syndrome and lepromatous leprosy.
CR1, in addition to having the four codominant size polymorph alleles, also exhibits Knops blood group polymorphism (Please fill in reference prop #107, now 2) and cis-acting Hind III restriction fragment length polymorphism (RFLP) (Please fill in reference prop #108, now 3). Little is known about the role of the Knops antigen in receptor function and therefore its role in autoimmune diseases, such as SLE. RFLP correlates with quantitative expression of CR1 on erythrocytes. RFLP has a 6.9 kilobase polymorph which correlates with low copy number expression of CR1 on erythrocytes. The other RFLP is 7.4 kilobases in size and correlates with high numeric expression. There is a controversy as to whether or not the 6.9 kilobase RFLP is found with increased frequency in SLE patients and their relatives. Similarly, controversy persists regarding the role of size polymorphism in genetic predisposition to SLE, autoimmune and pathogenic diseases.
The present invention is based on the discovery of a novel form of polymorphism in CR1. This novel polymorphism is exploited in a testing methodology which allows for early identification of individuals susceptible to diseases associated with CR1 function, offering the possibility of early and aggressive treatment in those patients.
The present invention is a system and method for correlating the ability of binding properties of human complement receptor (CR) and cellular susceptibility to a disease by identifying a CR genotype of a cell and quantifying complement protein binding by the cell expressing the CR genotype. Thereafter, the complement protein binding by the cell and complement protein binding by a second cell expressing a second Fcxcex3RI genotype is compared.
The present invention uses a single nucleotide polymorphism or combinations thereof within a CR genotype to identify individual susceptibility to a disease.
The methods of the present invention also extend to correlating the ability of a cell to bind complement protein and cellular susceptibility to a disease through identifying a Cr phenotype of a given cell and quantifying complement protein binding by said cell. Thereafter, complement protein binding by the cell is compared to a second cell having a second CR phenotype. In particular, single nucleotide polymorphisms are responsible for genotypical and phenotypical differences in CR herein.
The present invention further includes a commercial packaging including reagents for identifying single nucleotide polymorphisms in the CR genotype or phenotype of an individual as a test to identify individual susceptibility to a disease. The reagents further include instructions for the use thereof.